To see the interactions in real time, Suo and colleagues were the first to insert fluorescent markers into each of the four components of the enzyme called the palm, thumb, finger and little finger. They also placed markers in a strand of DNA. Previous studies have emphasized just one enzyme domain at a time.
The polymerase interacts with DNA to start the copying process in the presence of a nucleotide in this case, a molecule called dNTP. The scientists used fluorescence resonance energy transfer technology to see the action take place.
They described the process as a series of eight critical steps that proceed with three distinct movements in the presence of a dNTP. The enzyme stands alone until it binds to DNA. The enzyme and DNA undergo repositioning when the nucleotide binds to the polymerase, and the enzyme's domains begin to move, gripping the template DNA strand. The DNA strand must move from what is known as the pocket to allow binding and incorporation of the nucleotide in the correct place.
By step five in the process, the DNA copy has been extended, and in the following steps the enzyme begins its relaxation motions allowing the DNA to shift to allow for placement of the next nucleotide.
Those who study DNA polymerases seek to define what is called a rate-limiting event, which could be thought of as a moment when all parts of the process pause and check themselves over. Because errors occur in the DNA copying process, the rate-limiting step must be known to completely understand the copying mechanism, Suo noted.
To date, scientists have believed that the rate-limiting step in this process occurred long before the placement of the nucleotide. To check t
|Contact: Zucai Suo|
Ohio State University